The present invention relates to applications of an antibody directed to CX3CR1, an antibody directed to fractalkine and fractalkine.
Killer lymphocytes play a central role in elimination of pathogens that invade into living bodies, abnormal cancer cells and so forth. However, their excessive cytotoxic reactions sometimes destroy normal tissues and they are tightly associated with onsets of autoimmune diseases such as nephritis, rheumatism, diabetes mellitus and myocarditis. Killer lymphocytes are classified into NK cells, CD8T cells, CD4T cells, γδ T cells, NKT cells and so forth, which commonly have a mechanism for inducing apoptosis of a target cell. The most known mechanism is mediated by perforin, which is a pore-forming protein, and granzyme B, which is a serine protease, released from cytotoxic secretory granules of the killer lymphocytes. Perforin forms pores in target cells, and then granzyme B is mobilized from these pores into the target cells and induces apoptosis of the target cells. Further, FasL and TARIL belonging to the TNF family are expressed or secreted on the cell surface of killer lymphocytes, bound to a receptor on the target cells and induce apoptosis. The cytotoxic mechanism and the activation mechanism of killer lymphocytes have been studied in detail, but little is known about factors for regulating migration of these cells in living bodies.
Infiltration of lymphocytes from the bloodstream into an inflammatory tissue is considered to require multistep reactions involving cell adhesion molecules and cellular chemotactic factors. In a typical infiltration mechanism, an initial reaction is contact and rolling of lymphocytes on endothelial cells. These are mainly performed by adhesion molecules called selectin. By performing the rolling, the lymphocytes become able to sense cellular chemotactic factors locally produced and presented on vascular endothelial cells. The cellular chemotactic factors activate adhesion molecules called integrin mainly by signal transduction via a G protein-coupled seven-transmembrane receptor and induce strong adhesion between lymphocytes and endothelial cells. Then, finally, lymphocytes are infiltrated into a tissue through intercellular spaces of endothelial cells.
Chemokines are major cellular chemotactic factors in living bodies and regulate tissue infiltration of lymphocytes through acceleration of cell motility and activation of cell adhesion molecules. Chemokines are classified into four subfamilies, CC, CXC, C and CXXXC, depending on the sequence of their first two cysteine residues. Members of the CC, CXC and C chemokine subfamilies are secretory proteins composed of about 70 amino acids. Although they themselves do not have activity as adhesion molecules, they can induce cell adhesion. Secreted chemokines are bound to seven-transmembrane receptors on the surface of target cells and activate integrin through a trimer G protein to induce cell adhesion and migration. The cell-type specific activity of a chemokine is primarily determined by whether its specific receptor exists in a certain cell subset. Therefore, cell-type specificity of a chemokine bound to the receptor can be revealed by closely investigating an expression state of a specific chemokine receptor on lymphocytes for each cell subset.
Recently, in addition to the previously revealed cellular chemotactic mechanism, a novel simple lymphocyte infiltration mechanism was identified. This mechanism is mediated by fractalkine, which is expressed on activated endothelial cells, and a seven-transmembrane receptor, CX3CR1, which is expressed on monocytes, NK cells and a part of T cells in the bloodstream. Fractalkine is the only member of the CXXXC chemokine subfamily and has distinct structural and functional characteristics, which cannot be observed in other chemokines. Fractalkine is expressed on the cell surface as a membrane-bound chemokine which has a chemokine domain, a mucin domain, a transmembrane domain and an intracytoplasmic domain. When the membrane-bound fractalkine binds to CX3CR1, it can solely mediate strong adhesion without mediation by selectins or integrins even at a physiological blood flow rate. That is, the fractalkine-CX3CR1 cellular infiltration system mediates, by one-step reaction, the same function as that of the multistep cellular infiltration mechanism mediated by selectins and integrins. Further, secretory fractalkine secreted by shedding from the membrane-bound fractalkine binds to CX3CR1 and induces activation of integrin and cell chemotaxis as in the case of the known chemokines.
Further, expression of fractalkine is induced by treating vascular endothelial cells with inflammatory cytokines such as TNF or IL-1. CX3CR1 is expressed on monocytes, most of NK cells and a part of T cells, but not on neutrophils. Therefore, the fractalkine CX3CR1-cellular infiltration system is considered to be an extremely efficient mechanism for recruiting a kind of immunocytes onto endothelial cells of a damaged tissue or into the tissue. However, kinds of cells migrated by the cellular infiltration system and actions of the cellular infiltration system in inflammatory reactions have not been analyzed.